Although a relatively new technique, atomic force microscopy (AFM) has been proven a powerful structural tool in biology. However, despite the successes in acquiring high resolution surface structures of many macromolecules under ambient conditions, the ability of AFM for the study of large, individual complexes that are often exceedingly flexible is still very limited, primarily because of the deformation and damage caused by the scanning tip which exerts a fairly high pressure within the contact area between the tip and the sample. As a result, even with very sharp tips, the resolution has been relatively low on large structures. To overcome this problem and to expand the ability of AFM for the elucidation of molecular structures at the single molecule level, we have developed the first AFM that is operated at the liquid nitrogen temperature under ambient pressure (cryo-AFM). Our results clearly demonstrate that at cryogenic temperatures, the molecular rigidity is significantly improved, allowing clear and high-resolution images to be obtained reliably and reproducibly. With this novel technique, we have been able to reveal structural conformations for several macromolecules that were not known previously. In this continuation application, we seek to continue the development of this new technology for applications in structural biology, with a particular emphasis on single, flexible complexes. We will construct a multi-functional integrated system that will include an array of specimen preparation capabilities. We will also push the technology to improve its resolving power on well preserved, quickly frozen specimens (after deep etching). To achieve this goal, in addition to instrumental improvements, we also plan to utilize the newly developed nanotube tips and "single atom" probes in our cryo-AFM. As an integral part of this project, we will also apply this method to several carefully selected problems, including the structure of IgM, protein distribution in a cell membrane, the surface structure of the nuclear pore complex and an enveloped virus. These applications will provide the initial validation of the methodology, but at the same time, should also allow us to obtain critical information about these structures that have been difficult to study at the moment.